J. Agric. Food Chem. 1997, 45, 4838−4844
4838
Evidence That Maize Acetyl-Coenzyme A Carboxylase Does Not Function Solely as a Homodimer† Bev J. Incledon and J. Christopher Hall* Department of Environmental Biology, University of Guelph, Guelph, Ontario N1E 2W1, Canada
Acetyl-coenzyme A carboxylase (EC 6.4.1.2; ACCase) was purified from etiolated maize coleoptiles using cyclohexanedione affinity and DyeMatrex gel orange A, followed by anion-exchange chromatography. Purification yielded ACCase240, which was bound to orange A dye (purified 68×), and ACCase220, having no affinity for orange A dye (purified 79×). ACCase220 contained two biotinylated polypeptides with molecular mass of 220 and 85 kDa. The 85-kDa protein was separated from the 220-kDa protein and had ACCase activity. ACCase220 was composed of seven proteins as determined by native PAGE. Two-dimensional electrophoresis (native/SDS PAGE) of ACCase220 revealed that these seven native proteins share constituent subunits. These subunits of ACCase220 were cross-linked using dithiobis(succinimidylpropionate), and unassociated polypeptides were removed by YM100 ultrafiltration. Reduction of cross-linked ACCase resulted in the reappearance of multiple polypeptides originally observed by SDS PAGE. On the basis of these results, we provide the first evidence that gramineae ACCase does not function solely as a homodimer. Keywords: ACCase; quaternary structure; enzyme complex; chemical cross-linking; graminicide INTRODUCTION
Acetyl-coenzyme A carboxylase (EC 6.4.1.2; ACCase) is the first dedicated enzyme in the de novo fatty acid biosynthetic pathway and the target site for two commercial classes of grass-selective herbicides, the cyclohexanediones (CHDs) and (aryloxy)phenoxypropionates (AOPPs) (Secor and Cseke, 1988; Rendina et al., 1989; Burton et al., 1991). These two classes of herbicides have been shown to selectively inhibit ACCase of gramineae weeds and are therefore used for their control in broadleaf crops (Betts et al., 1992; Devine et al., 1992; Gronwald et al., 1992; Tardif et al., 1993). It has been hypothesized that inhibition of ACCase is not the sole mode of action of these herbicides (Shimabukuro and Hoffer, 1996); however, it has been demonstrated that there is a specific interaction of these herbicides with ACCase. Furthermore, AOPP- and CHD-resistant and susceptible grasses show differential levels of ACCase sensitivity to these herbicides (Walker et al., 1990; Tardif and Powles, 1994; Herbert et al., 1996). ACCase catalyzes the ATP-dependent conversion of acetyl-coenzyme A to malonyl-coenzyme A via a dual active-site mechanism (Finlayson and Dennis, 1983). Acetyl-coenzyme A carboxylation is achieved by way of two distinct half-reactions. The first reaction involves the hydrolysis of ATP and carboxylation of the biotin cofactor. The second half-reaction involves the transfer of the carboxyl group from the biotin cofactor to acetylcoenzyme A, resulting in the formation of malonylcoenzyme A (Finlayson and Dennis, 1983). To facilitate this reaction, the bicarbonate-derived carboxyl group must be shuttled from the first active site to the second active site. The overall reaction can be separated into * Corresponding author [telephone (519) 824-4120, ext. 2740; fax (519) 837-0442; e-mail jchall@ evbhort.uoguelph.ca]. † This work was supported by a Research Grant from Agriculture Canada/NSERC and DowElanco provided to J.C.H. B.J.I. is a recipient of an NSERC post graduate scholarship (PGS-B). S0021-8561(97)00479-2 CCC: $14.00
three distinct enzymatic functions: biotin carboxylation, carboxyl transfer, and acetyl-CoA carboxylation. The ACCases of dicotyledonous plants exist as a multimeric enzyme complex (Kannangara and Stumpf, 1972; Wurtele and Nikolau, 1992; Alban et al., 1994; Konishi and Sasaki, 1994; Roesler et al., 1994, 1996; Konishi et al., 1996). Furthermore, Konishi et al. (1996) states that all monocotyledonous plants with the exception of the gramineae also have a multisubunit quaternary structure. The multimeric quaternary arrangement is similar to the prokaryotic system where the three enzymatic functions reside on separate, constituent proteins (Kannangara and Stumpf, 1972; Wurtele and Nikolau, 1992; Alban et al., 1994; Konishi and Sasaki, 1994; Roesler et al., 1994, 1996; Konishi et al., 1996). A homodimeric form also exists in many dicotyledonous and monocotyledonous plants. For example, Alban et al. (1994) reported that pea ACCase exists in two forms, the predominant form being the multimeric complex and the minor form being homodimeric. Furthermore, Dehaye et al. (1994) reported that the two forms of ACCase in pea have differential sensitivity to the AOPP and CHD herbicides, with the multimeric form being resistant and the homodimeric form being susceptible to these herbicides. Currently, it is believed that gramineae species have only a homodimeric form of ACCase which is sensitive to AOPPs and CHDs, thereby imparting selectivity between gramineae and dicotyledonous species (Egli et al., 1993; Herbert et al., 1996; Konishi et al., 1996). In several grass species, it has been reported that isozymes of homodimeric ACCase exist and that these isozymes exhibit differential sensitivity to the AOPP and CHD herbicides (Egli et al., 1993; Konishi et al., 1996). However, Nikolau and Hawke (1984) reported that maize ACCase was composed of six 60-61-kDa constituent biotinylated proteins. Similarly, Evenson et al. (1994) reported that Lolium multiflorum ACCase might have an associated protein or another subunit that does not contain biotin. Furthermore, gramineae ACCase has not yet been purified to homogeneity, and consequently, no convincing evidence for the presence of a © 1997 American Chemical Society
Evidence of Multimeric ACCase in Maize
solely homodimeric quaternary arrangement has been presented. Low molecular mass (
